Screening method for gene variation

ABSTRACT

A method for screening presence or absence of a variation in a region of a nucleic acid which comprises the steps of (a) preparing a sample containing a test nucleic acid corresponding to the region, (b) preparing a probe having a base sequence fully complementary to a normal sequence of the region, and a plurality of probes each having at least one base not complementary to the normal sequence, (c) fixing the probes in separate regions on a surface of a substrate to prepare a DNA array substrate, (d) reacting the test nucleic acid with the probes on the DNA array substrate, (e) measuring signals in each region where the signals are originated from respective hybrids formed between the test nucleic acid and one of the probes, and (f) calling variation in the test nucleic acid using a pattern of total signals of all regions.

This application is a division of application Ser. No. 09/942,588, filedAug. 31, 2001, which is incorporated herein by reference.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates to a variation detection method and asystem for it useful for screening of a gene variation etc.

2. Related Background Art

One of the techniques for sequencing nucleic acid etc. or for detectingthe sequence is to utilize a DNA array. U.S. Pat. No. 5,445,934discloses a DNA array where 100,000 or more oligonucleotide probes arebonded in 1 inch square. Such a DNA array has an advantage that manycharacteristics can be examined at the same time with a very smallsample amount. When a fluorescence-labeled sample is poured onto such aDNA chip, DNA fragments in the sample bind to probes having acomplementary sequence fixed on the DNA chip, and only that part can bediscriminated by fluorescence to elucidate the sequence of the DNAfragment in the DNA sample.

Sequencing By Hybridization (SBH) is a method for examining the basesequence utilizing such a DNA array and the details are described inU.S. Pat. No. 5,202,231. In the SBH method, all possible sequences of anoligonucleotide of a certain length are arranged on the substrate, thenfully matched hybrids formed by hybridization reaction between probesand the sample DNA are detected. If a set of fully matched hybrids isobtained, the set will give an assembly of overlapping sequences withone base shift being a part of one certain sequence, which sequence isextracted for calling.

Including the SBH method, when complementariness between anoligonucleotide and a sample DNA is examined, it is very difficult tocall whether a hybrid was formed or not using one probe for one testitem, since the stability of a hybrid differs sequence to sequence, andthere is no perfect signal for calling the full complementariness.Science vol. 274 p. 610-614, 1996 discloses a method for calling bycomparing the signal intensity of a perfect match hybrid and the weakerintensities of one-base mismatch hybrids. In this method, 15-meroligonucleotide probes, differing from each other only by onemismatching base at the center of the sequence, are prepared, and thefluorescence intensities of the hybrids of the probes are compared. Whenthe intensity of the full matched hybrid is stronger than that of otherhybrids by a predetermined rate, it is called positive.

Further, U.S. Pat. No. 5,733,729 discloses a method using a computer todifferentiate a base sequence of a sample from a comparison offluorescence intensities of obtained hybrids for more accurate calling.

However, the actual binding strength of a hybrid depends on the GCcontent etc., and difference of the fluorescence intensity between afull match hybrid and a one-base mismatch hybrid also varies in aconsiderable range depending on the sequence. Thus, a method for callingwhether a sequence is fully complementary to a probe or not, using a 15mer oligonucleotide probe to compare it with other three probes havingone mismatched base at the center thereof, can provide more accuracy ifeach stability is evaluated theoretically or empirically beforecomparison.

In addition, accurate calling requires precise quantification ofsignals, and therefore, precision apparatuses such as a confocal lasermicroscope. Furthermore, in order to measure the fluorescence intensityof a hybrid of every probe and to determine the gene sequence byanalyzing the data, a large-scale computer apparatus as well as adetection apparatus for reading the arrays are further required.Therefore, this is a big obstacle for ready use of the DNA array.

On the other hand, gene diagnosis using such a DNA array may be used ingroup medical examination, individual gene examination orgene-polymorphism study. In such a case, however, the above describedprecise measurement and analysis are not always required, where a largeamount of samples are rapidly treated at a low cost in order to find outvariated samples concerning a specific item from a large number ofnormal samples. Further, the precision apparatus and analysis asdescribed above will be expensive. Accordingly, a concept that screeningof the presence or absence of a variation is first performed, and then,detailed examinations of the samples suspected of variation are carriedout by screening, saving both time and cost.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method suitable formass screening so as to determine rapidly the presence or absence of agene variation without need of an expensive apparatus and a complexanalysis.

The present invention provides a DNA array in which a group of probeswhich will give strong signals forming hybrids with a normal genesequence, and a group of probes having sequences expected to formhybrids with gene variants are separately arranged, on the premise thatthe base sequence of a normal gene and those of variants have alreadybeen established. Furthermore, the above described object is achieved byproviding a detection method using such an array.

According to one aspect of the present invention, there is provided amethod for screening of the presence or absence of variation in a regionof a nucleic acid comprising the steps of:

-   -   (a) preparing a test nucleic acid corresponding to the region;    -   (b) preparing a probe having a base sequence fully complementary        to a normal sequence of the region, and a plurality of probes        each having at least one base not complementary to the normal        sequence;    -   (c) fixing the probes in separate regions on a surface of a        substrate to prepare a DNA array substrate;    -   (d) reacting the test nucleic acid with the probes on the DNA        array substrate;    -   (e) measuring signals in each region totally where the signals        are originated from respective hybrids formed between the test        nucleic acid and one of the probes; and    -   (f) determining the presence or absence of mutation in the test        nucleic acid comparing with a histogram pattern of signals of        all regions obtained using a normal sample without variation.

According to another aspect of the invention, there is provided a DNAarray substrate for screening a variation in a region of a nucleic acid,wherein

-   -   a full match probe fully complementary to a normal sequence of        the region, and a plurality of mismatch probes having at least        one base mismatch to the sequence are arranged on the substrate;        and    -   the probes are arranged to form at least two separate regions        selected from:    -   a first region containing at least one probe which provides a        signal of a certain intensity on reaction with a nucleic acid        having the normal sequence,    -   a second region containing at least one probe which provides a        weaker signal than the probe of the first region on reaction        with a nucleic acid having normal sequence, and    -   the third region containing at least one probe which provides no        signal on reaction with a nucleic acid having normal sequence.

According to still another aspect of the present invention, there isprovided a system for detecting variation comprising a DNA arraysubstrate as described above and a signal measuring apparatus whichmeasures signals from separate regions of the DNA array substrate.

The present invention can provide a method suitable for mass screening,so as to rapidly determine only the presence or absence of a genevariant, without need of an expensive apparatus and complex analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic example of arrangement of separate regions on aDNA array substrate, and FIG. 1B shows a histogram of fluorescenceintensities (pattern) corresponding to separate regions of the abovearrangement, measured using a line sensor:

FIG. 2 shows an arrangement of 64 types of DNA probes;

FIG. 3 shows a pattern of the fluorescence intensities obtained byhybridization;

FIG. 4 shows normal fluorescence intensities obtained by hybridizationwhere Gr. NO. denotes group number;

FIG. 5 is a distribution pattern of fluorescence intensitiescorresponding to the separate regions of the arrangement, measured usinga line sensor;

FIG. 6 is a distribution pattern of fluorescence intensitiescorresponding to the separate regions of the arrangement, measured usinga line sensor;

FIG. 7 is a distribution pattern of fluorescence intensitiescorresponding to the separate regions of the arrangement, measured usinga line sensor;

FIG. 8 is a distribution pattern of fluorescence intensitiescorresponding to the separate regions of the arrangement, measured usinga line sensor; and

FIG. 9 shows normal fluorescence intensities obtained by hybridizationwhere Gr. NO. denotes group number.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a screening method for gene variantsusing a DNA array in which a group of probes which will give strongsignals forming hybrids with a normal gene sequence, and a group ofprobes having sequences expected to form hybrids with gene variants areseparately arranged, on the premise that the base sequence of a normalgene and those of variants have already been established. Furthermore,the above described object is achieved by providing a detection methodusing such an array.

Here, the present invention will be described in detail with exampleswhere signals from the separate regions are fluorescence. However,signals in the present invention are not limited to fluorescence but maybe other light signals or electric signals.

Binding strength between single-stranded nucleic acids to form a hybridis controlled by various factors, and when a probe having a length ofabout 12 mer to 25 mer is used, it is practically difficult to perfectlyexclude hybrids having one-base mismatches.

When the signal of the hybrid to be detected is a light such asfluorescence, the following phenomena are observed. Fluorescencestability of a hybrid having two mismatches (two-base mismatch hybrids)is much lower than that of the one-base mismatched hybrid, regardless ofpositions, continuity or discontinuity of the mismatched bases. On theother hand, signal from a three-base mismatch hybrid is hardly observed.However, one-base mismatch hybrids may have more than 50% signalintensity of a full match hybrid. Thus, when a sample is a nucleic acidof normal sequence, strong fluorescence is observed in a region where afull match probe and probes having one mismatching base have beenarranged, while the fluorescence intensity in a region where hybrids oflow stability are formed is almost zero. On the other hand, when asample is a nucleic acid of a variant sequence, the probe fullycomplementary to the normal sample makes a mismatch hybrids with thesample. Thus the fluorescence is weaker at a region than that in case ofthe normal sample, at the same time, fluorescence from a full matchhybrid and one-base mismatch hybrids with the sample appears in anotherregion where low signals are expected in case of the normal nucleicacid. Accordingly, by comparing fluorescence histogram of the regions,it is possible to distinguish between the normal nucleic acid and thevariant nucleic acid.

In the present invention, a DNA array substrate where probes arearranged in separate regions according to the fluorescence intensitiesof their hybrids with a normal nucleic acid is used for more accuratecalling. First, hybridization reaction of the normal nucleic acid witheach probe is performed, and based on the fluorescence intensities ofthe hybrids obtained, separate regions each containing probescorresponding to strong fluorescence, no fluorescence, and moderate(weak) fluorescence are located at predetermined positions on thesubstrate.

When performing a test on multiple items simultaneously, the substrateshould be divided into areas for respective items. In each area, probeshaving full match and one-base mismatch sequences to the normal genesequence are arranged in a region, which are expected to give highsignals, and the other probes having more than two-base mismatch arearranged in a separate region depending on the items. Thus we candiscriminate normal test samples from variated ones.

The arrangement can be determined according to the type of the sensor tobe used. For example, when a line sensor is used, each region isarranged from the left to right in the substrate in order of thestrength of fluorescence obtainable by hybridization with the normalnucleic acid. Thus, the fluorescence intensity will be maximum in theleft, then gradually decrease and come to zero in several regions in theright of the substrate. When an area sensor is used, it is necessary toevaluate the fluorescence quantity of at least two separate regionscontaining a group of probes which will provide the maximum fluorescenceand a group of probes which will not provide any fluorescencerespectively.

This will be described more specifically.

In the present invention, for example, whether a gene is normal or notcan be called by providing a region containing a probe that forms ahybrid with a normal nucleic acid and the other region containing probesthat form hybrids with variant genes separately on a DNA array, andtaking a ratio of the signal from a hybrid corresponding to the normalnucleic acid and to the signals from hybrids corresponding to variantgenes.

However, because one-base mismatched hybrids sometimes have strongsignals, it is difficult to detect variation in the test sample when aDNA array where only probes being full match or one-base mismatch to anormal sequence are arranged is used. It is important that probes havingtwo-base mismatch to the normal nucleic acid is present in the DNAarray, which might be one-base mismatch probes to the test sample toform hybrids having strong signals.

Therefore, in the present invention, in order to judge more accurately,a preferable method is as follows. Probes having full match and one-basemismatch sequences to the normal nucleic acid sequence, which mostsamples in mass screening have, are arranged in a specified region on aDNA array substrate. In addition, probes having two- or three-basemismatch are arranged in a region different from the above region forhigh stability hybrids. Then, a hybridization reaction is performed withthe normal nucleic acid or a sample, using the DNA array substrate ofsuch an arrangement. Then the total of signals for each separate regionis measured, and a pattern obtained with a sample nucleic acid iscompared that with the normal nucleic acid are compared to determine thepresence or absence of variation.

For example, when 64 probes (4×4×4=64) where different bases arearranged at three positions in 18 base length (Table 1, 5′-terminus ison the left) are used supposing that variation occurs at these threepoints, for any sample there should be present a probe fullycomplementary to the sample, nine one-base mismatch probes, 27 two-basemismatch probes and 27 three-base mismatch probes. Furthermore, it isrelatively easy to set conditions of the hybridization reaction suchthat fluorescence is observed with the full match and one-base mismatchhybrids but not with three-base mismatch hybrids. Some two-base mismatchprobes form hybrids and others not, depending to their sequences. TABLE1 SEQ ID NO: Sequence 1 GATGGGACTCAAGTTCAT 2 GATGGGACTCAGGTTCAT 3GATGGGACTCACGTTCAT 4 GATGGGACTCATGTTCAT 5 GATGGGACTCGAGTTCAT 6GATGGGACTCGGGTTCAT 7 GATGGGACTCGCGTTCAT 8 GATGGGACTCGTGTTCAT 9GATGGGACTCCAGTTCAT 10 GATGGGACTCCGGTTCAT 11 GATGGGACTCCCGTTCAT 12GATGGGACTCCTGTTCAT 13 GATGGGACTCTAGTTCAT 14 GATGGGACTCTGGTTCAT 15GATGGGACTCTCGTTCAT 16 GATGGGACTCTTGTTCAT 17 GATGGGGCTCAAGTTCAT 18GATGGGGCTCAGGTTCAT 19 GATGGGGCTCACGTTCAT 20 GATGGGGCTCATGTTCAT 21GATGGGGCTCGAGTTCAT 22 GATGGGGCTCGGGTTCAT 23 GATGGGGCTCGCGTTCAT 24GATGGGGCTCGTGTTCAT 25 GATGGGGCTCCAGTTCAT 26 GATGGGGCTCCGGTTCAT 27GATGGGGCTCCCGTTCAT 28 GATGGGGCTCCTGTTCAT 29 GATGGGGCTCTAGTTCAT 30GATGGGGCTCTGGTTCAT 31 GATGGGGCTCTCGTTCAT 32 GATGGGGCTCTTGTTCAT 33GATGGGCCTCAAGTTCAT 34 GATGGGCCTCAGGTTCAT 35 GATGGGCCTCACGTTCAT 36GATGGGCCTCATGTTCAT 37 GATGGGCCTCGAGTTCAT 38 GATGGGCCTCGGGTTCAT 39GATGGGCCTCGCGTTCAT 40 GATGGGCCTCGTGTTCAT 41 GATGGGCCTCCAGTTCAT 42GATGGGCCTCCGGTTCAT 43 GATGGGCCTCCCGTTCAT 44 GATGGGCCTCCTGTTCAT 45GATGGGCCTCTAGTTCAT 46 GATGGGCCTCTGGTTCAT 47 GATGGGCCTCTCGTTCAT 48GATGGGCCTCTTGTTCAT 49 GATGGGTCTCAAGTTCAT 50 GATGGGTTCTAGGTTCAT 51GATGGGTCTCACGTTCAT 52 GATGGGTCTCATGTTCAT 53 GATGGGTCTCGAGTTCAT 54GATGGGTCTCGGGTTCAT 55 GATGGGTCTCGCGTTCAT 56 GATGGGTCTCGTGTTCAT 57GATGGGTCTCCAGTTCAT 58 GATGGGTCTCCGGTTCAT 59 GATGGGTCTCCCGTTCAT 60GATGGGTCTCCTGTTCAT 61 GATGGGTCTCTAGTTCAT 62 GATGGGTCTCTGGTTCAT 63GATGGGTCTCTCGTTCAT 64 GATGGGTCTCTTGTTCAT

When these 64 probes are grouped into every eight probes in order of thefluorescence intensity obtained by hybridization with the normal nucleicacid (hereinafter “fluorescence intensity of a probe(s)” means expectedintensity of a hybrid of the probe with a nucleic acid of normalsequence, if not otherwise stated), the total fluorescence intensity ofthe first group should be extremely high and the total fluorescencequantity of the sixth, seventh and eighth groups should be zero. Suchclassification by the fluorescence intensity may be performedempirically or theoretically through calculation.

Then, the hybridization reaction is performed in an actual system andthe total quantity of fluorescence is determined for each classifiedgroup. Particularly, the fluorescence quantities of the sixth, seventhand eighth groups are important. Normally, fluorescence is not expectedfor these groups. When fluorescence is detected unexpectedly for thesegroups, it is understood that the sample is not normal but havingvariation at a part of the sequence. When the fluorescence is notdetected for these groups, most of the sequences are normal.

For more accuracy, however, it is necessary to compare the fluorescencequantity of the first group with a normal case. When the fluorescencequantity is significantly lower than that expected for normal sequence,base variation is suspected. Further the histogram of the fluorescenceintensity in the regions having medium fluorescence intensity, i.e. thesecond, third and fourth groups, should be compared with that of thenormal one.

For more effective detection of variation, one may consider a methodwherein the probes are arranged in order of signal intensity, that is,lined in the order of sequences of full match, one-base mismatch,two-base mismatch, and three-base mismatch to the normal sequence fromone end, so that signal intensity may be zero in the region opposite tothe region where the full match probe is placed. In this case, the totaldistribution pattern of the fluorescence intensity is examined. Forexample, when using an arrangement of the separate regions shown in FIG.1A (one column corresponds to one separate region), a monotonouslydecreasing pattern as shown in FIG. 1B is obtained for the normal casewhile patterns having peaks in the intermediate regions will be obtainedwith variants.

The above arrangement method is similarly used for the case where theprobes for multi-item testing are arranged on the same substrate. Foreach item, first, the full matched probe (to the normal sequence), thenone-base mismatched sequences, two-base mismatched sequences and so onare placed in order of the strength of fluorescence intensity expected.

Such concept is universally applicable to any number of variation, notlimited to the above method where the variation for only three bases istested.

In addition, while we explained the cases where the signals can beobtained when the hybrids are formed, the method may be set such thatsignals are not obtained when the hybrids are formed, and obtained whenthe hybrids are not formed, depending on the signal generating system.

The sample nucleic acid can be prepared by extracting from the gene tobe tested according to the necessity. The control normal nucleic acidcan be synthesized on the basis of the known sequence.

The length of the probe fixed to the substrate is not limited so long asit is suitable for detection, for example, preferably 8 mer to 30 mer,more preferably 12 mer to 25 mer.

Probes may be fixed to the substrate by various methods, but dropletapplication by the ink jet method is preferably used in order to arrangespots of fixed probes with high efficiency, high density and high speed.

Each spot in the separate region is preferably 70 to 100 μm in diameterand spaced not to connect each other.

The spot number in each region is set so that the spots expected to havehigh fluorescence intensity can be measured together and the spotsexpected to have no fluorescence can be measured together, consideringthe constitution of the sensor.

A system for detecting variation of the present invention comprises aDNA array substrate wherein plural separate regions are arranged in aprescribed arrangement, and a sensor for measuring the signals in theseparate regions subjected to measurement. In addition, for calling thepresence or absence of variation using the DNA array substrate where theplural separate regions are provided, the signals from all separateregions may be detected and the results are used for calling thepresence or absence of variation or, as a simpler method, signals may becompared between certain regions selected so as to detect the presenceor absence of variation.

Computer analysis is conveniently performed connecting a computer systemto the detection system. For many variants of a gene sequence, histogrampatterns of signal intensity are prepared and stored in the computer,which helps the determination of variant easily and correctly.

As the sensor for signal detection, different types of photodiodes (e.g.Hamamatsu Photonics K.K.-made) are used. For example, some of thedivided type silicon photodiode arrays can be used as both line sensorand area sensor. Particularly, those having a photo-receiving face of 1to 2 mm×(2 to 3) mm are suitably used.

EXAMPLES

The present invention will be described in more detail referring toExamples below. Herein, “%” means “weight %”.

Example 1

Preparation of DNA Array for Detection of Variant Gene for Line Sensor

1) Preparation of DNA Array Linked with 64 Types of Probes

(1) Probe Design

It is well known that in the base sequence CGGAGG corresponding to theAA248 and AA249 of the tumor suppressor gene p53, frequently observedvariation is those the first C to T, the second A to G for AA248, andthe third G to T for AA249. Accordingly, aiming at these threepositions, 64 types of probes were designed.

That is, the designed nucleic acid are 18-mer nucleic acids harboringvariegated above mentioned six bases sandwiched between the commonsequences, represented by 5′ATGAACNNGAGNCCCATC3′ (SEQ ID NO: 68) where Ncorresponds to any of 4 bases, A, G, C and T. Actual probes to detectthe above sequence should be have a complementary sequence of5′GATGGGNCTCNNGTTCAT3′ (SEQ ID NO: 69).

FIG. 2 shows an arrangement of 64 types of DNA probes on a substrate. Asequence 5′ ATGAACCGGAGGCCCATC3′ (SEQ ID NO: 65) corresponding to thenormal gene is expected to form a hybrid with DNA of probe 42 of5′GATGGGCCTCCGGTTCAT3′ (SEQ ID NO: 42) positioned at the third from theright and the third from the top.

(2) Preparation of Substrate Introduced with Maleimide Group

Substrate Cleaning

A glass plate of 1 inch square was placed in a rack and soaked in anultrasonic cleaning detergent overnight. Then, after 20 min ofultrasonic cleaning, the detergent was removed by washing with water.After rinsing the plate with distilled water, ultrasonic treatment wasrepeated in a container filled with distilled water, for additional 20min. Then the plate was soaked in a prewarmed 1N sodium hydroxidesolution for 10 min, washed with water and then distilled water.

Surface Treatment

Then the plate was soaked in an aqueous solution of 1% silane couplingagent (product of Shin-Etsu Chemical Industry: Trade name KBM 603) at aroom temperature for 20 min, thereafter nitrogen gas was blown on theboth sides blowing off water to dryness. The silane coupling treatmentwas completed by baking the plate in an oven at 120° C. for 1 hour.Subsequently, 2.7 mg of EMCS (N-(6-maleimidocaproyloxy) succinimide:Dojin Company) was weighed and dissolved in a 1:1 solution ofDMSO/ethanol (final concentration: 0.3 mg/ml). The glass substratetreated with the silane coupling agent was soaked in this EMCS solutionfor 2 hours to react the amino group of the silane coupling agent withthe succimide group of EMCS. At this stage, the maleimide group of EMCSis transferred to the glass surface. After that, the glass plate waswashed with ethanol, and dried with nitrogen gas to be used for acoupling reaction with DNA.

3. Coupling of DNA to the Substrate

Synthesis of 64 DNA Probes

The 64 types of probe DNAs shown in Table 1 each having an SH group(thiol group) at the 5′ terminus were synthesized by a standard method.

Ejection of DNA Probes

Each DNA was dissolved in water and diluted with SG Clear (aqueoussolution containing 7.5% of glycerin, 7.5% of urea, 7.5% of thiodiglycoland 1% of acetylenol EH), to a final concentration of 8 μM.

Then 100 μl of this DNA solution was filled into a nozzle of a BJprinter Head BC 62 (Canon) modified to eject a small amount, and toeject six solutions per head. Two heads were used at a time so that 12types of DNAs could be ejected at once, and the heads were changed 6times so that 64 spots of 64 types of DNAs were formed independently onthe predetermined positions. Thus obtained was a DNA array in whichseparate regions were arranged in a predetermined manner. The pitch ofspots was 200 μm and the area formed with 8×8 spots was about 2 mm×2 mm.

After that, the plate was left standing in a humidified chamber for 30min for linking reaction of the probe DNA to the substrate.

2. Measurement of Fluorescence Intensity of 64 Hybrids with Normal p53Sequence.

(1) Hybridization Reaction

Blocking Reaction

After completion of the reaction, the substrate was washed with a 1 MNaCl/50 mM phosphate buffer solution (pH 7.0) to wash out thoroughly theDNA solution on the glass surface. Then, this was soaked in an aqueoussolution of 2% bovine serum albumin and allowed to stand for 2 hours tocarry out a blocking reaction.

Preparation of Model Sample DNA

Rhodamine labeled DNA No. 1 (SEQ ID NO: 65) of the same length as theprobes but having the normal sequence of p53 gene was prepared. Thesequence is shown below and rhodamine is bonded to the 5′ terminus.(Synthesis of model sample of DNA).

The labeled DNA No. 65 (single strand) having the normal sequence of p53gene (complementary to No. 42) and the same length in the same region asthe probe DNA was prepared. The sequence is as shown below whererhodamine (Rho) is bound to the 5′-terminal. No. 65:5′-Rho-ATGAACCGGAGGCCCATC-3′ (SEQ ID NO: 65)Reaction Condition of Hybridization

Two milliliters of 50 nM DNA solution of a model sample containing 100mM NaCl was placed into a container containing the DNA array substratefor a hybridization reaction. Initially it was heated at 70° C. for 30min, then the temperature of the incubator was lowered to 40° C. and thereaction was continue for 3 hours.

(2) Detection

Method

The detection was performed by connecting an image analysis processingapparatus, ARGUS (a product of Hamamatsu Photonics) to a fluorescencemicroscope (a product of Nicon).

(3) Result

Distribution of the fluorescence quantity on the substrate obtained isshown in FIG. 3.

Example 2

Preparation of DNA Array for Detection of Variant Gene for Line Sensor

(1) Preparation of DNA Array for Detection of Variation

In Tables 2 and 3, these 64 probes are grouped in every 8 probes inorder of intensity based on the above described results. Thefluorescence intensity of the first group should be extremely strong,and the total fluorescence quantity of the sixth, seventh and eighthgroups should be zero. TABLE 2 Group SEQ ID No. NO: Type of Mismatch 142 Full mismatch 58 One-base mismatch 34 One-base mismatch 41 One-basemismatch 46 One-base mismatch 10 One-base mismatch 44 One-base mismatch43 One-base mismatch 2 26 One-base mismatch 38 One-base mismatch 50Two-base mismatch 2 Two-base mismatch 6 Two-base mismatch 9 Two-basemismatch 11 Two-base mismatch 12 Two-base mismatch 3 14 Two-basemismatch 18 Two-base mismatch 22 Two-base mismatch 25 Two-base mismatch27 Two-base mismatch 28 Two-base mismatch 30 Two-base mismatch 33Two-base mismatch 4 35 Two-base mismatch 36 Two-base mismatch 37Two-base mismatch 39 Two-base mismatch 40 Two-base mismatch 45 Two-basemismatch 47 Two-base mismatch 48 Two-base mismatch

TABLE 3 Group SEQ ID No. NO: Type of Mismatch 5 54 Two-base mismatch 57Two-base mismatch 59 Two-base mismatch 60 Two-base mismatch 62 Two-basemismatch 1 Three-base mismatch 3 Three-base mismatch 4 Three-basemismatch 6 5 Three-base mismatch 7 Three-base mismatch 8 Three-basemismatch 13 Three-base mismatch 15 Three-base mismatch 16 Three-basemismatch 17 Three-base mismatch 19 Three-base mismatch 7 20 Three-basemismatch 21 Three-base mismatch 23 Three-base mismatch 24 Three-basemismatch 29 Three-base mismatch 31 Three-base mismatch 32 Three-basemismatch 49 Three-base mismatch 8 51 Three-base mismatch 52 Three-basemismatch 53 Three-base mismatch 55 Three-base mismatch 56 Three-basemismatch 61 Three-base mismatch 63 Three-base mismatch 64 Three-basemismatch

Then, the surface of the substrate was separated into eight columns toarrange the first group, the second group and so on in order ofintensity from the left to the right. Then, the probes were arranged atthe positions of the corresponding probe numbers as shown in FIG. 4using the ink jet method in the same manner is in Example 1 to preparethe DNA arrays for detecting variation. The lines composed of each grouphave intervals of 200 μm.

(2) Testing Normal Gene Using DNA Array

The hybridization reaction was carried out under the same conditions asin Example 1. Thereafter, the total fluorescence of each group wasdetected using a line sensor (S 272102: Hamamatsu Photonics K.K.-made).

The results are shown in FIGS. 4 and 5. The fluorescence of the firstgroup was the highest and the intensity of the second group decreasesbelow the half of that. Fluorescence was hardly observed in the sixth,seventh and eighth groups.

Example 3

Detection of Variant Gene Using DNA Array (1)

1. Synthesis of Model Variant DNA

The labeled DNA No. 66 having the same length as the probes and asequence complementary to No. 46 probe that differs by one base from thenormal sequence of p53 gene was prepared. The sequence is shown below.Rhodamine was bound to the 5′-terminal. The underlined part is thevariant position. No. 66: 5′-Rho-ATGAACCAGAGGCCCATC-3′ (SEQ ID NO: 66)Reaction Conditions for Hybridization

The hybridization reaction was carried out under the same conditions asin Example 1. The concentration of the model sample DNA was 50 nM.

Detection by Line Sensor

After the hybridization reaction, the DNA array was evaluated in thesame manner as in Example 2 using a line sensor. As shown in FIG. 6, thefluorescence was observed in the sixth, seventh and eighth groups wherethe fluorescence was not observed with the normal gene. Thus, it waseasily judged that the sample gene was not normal. Further, since thefluorescence intensity was high at the first and the fourth groupscorresponding to probe Nos. 35 to 48, it is inferred that they have thevariation in the upper right quarter of FIG. 2 with a sequence veryclose to the normal gene.

This result shows that the screening method for variant genes of thepresent invention is extremely effective.

Example 4

Detection of Variant Gene Using DNA Array (2)

The hybridization reaction was carried out under the same conditions asin Example 3, except that the concentration of the model target geneused for the hybridization reaction was changed to 5 nM. The result isshown in FIG. 7.

Since the results similar to Example 3 were obtained to show that themethod of the present invention works not depending on the hybridizationreaction conditions.

Example 5

Detection of Variant Gene Using DNA Array (3)

Synthesis of Variant Model Sample DNA

The labeled DNA No. 67 having the same length as the probes and asequence complementary to No. 10 probe that differs by one base from thenormal sequence of p53 gene was prepared. The sequence is as shown belowwhere Rho represents rhodamine bound to the 5′-terminus. The underlinedpart is the variant position. No. 67: 5′-Rho-ATGAACCGGAGTCCCATC-3′ (SEQID NO: 67)Hybridization Reaction

The hybridization reaction was carried out under the same conditions asin Example 1 using the above variation model sample DNA. Theconcentration of the sample was 50 nM. The result is shown in FIG. 8.

Fluorescence was observed in the sixth and seventh groups which was notobserved for the normal gene, showing that this was not a normal gene.Since the fluorescence in the second group was higher than in the firstgroup which would be the highest for the normal gene, it is presumedthat it has a variation included in the probe sequences of the secondgroup (the upper left quarter in FIG. 2).

This result shows that the screening method for variant genes of thepresent invention is feasible not depending on the variation types.

Example 6

Preparation of DNA Array for Area Sensor

The probes grouped as shown in Example 2 were arranged by the ink jetmethod in the separate regions on the substrate as shown in FIG. 9 toprepare the DNA array for area sensor.

Then, the hybridization reaction was performed using the variant modelsample used in Example 4. As a result, the fluorescence was observed inthe sixth, seventh and eighth groups which was not observed for thenormal gene, therefore, it was easily judged that this gene was avariant one.

1. A method for screening of the presence or absence of variation in aportion of a sample nucleic acid comprising the steps of: (a) providinga DNA array substrate by: i) preparing a substrate; ii) preparing agroup of probes, each probe having a base sequence that hybridizes witha wild-type sequence of the portion to give a strong signal; iii)preparing a group of probes, each probe having a base sequence that isexpected to hybridize with a gene variant but not with the wild-typesequence to give a strong signal, the group not containing any probes ofthe group prepared in ii); and iv) fixing each probe on the substrate asa separate probe spot in such a manner that probe spots of each groupare arranged in one region on the substrate different from a region inwhich the probe spots of other groups are arranged and the probe spotsare grouped such that each said region contains probes not found inother regions; (b) reacting the sample nucleic acid with the probes onthe DNA array substrate; (c) measuring a signal intensity of each regionas a total of signals originating from respective hybrids formed betweenthe sample nucleic acid and the probes to obtain a histogram pattern ofsignal intensity of the regions; and (d) determining the presence orabsence of mutation in the sample nucleic acid comparing with thehistogram pattern with a histogram pattern obtained using an arraysubstrate obtained by step (a) and a reference nucleic acid having thewild-type sequence.
 2. The method according to claim 1, wherein thesignal is a light and a total light quantity emitted from each region ismeasured as the signal intensity.
 3. The method according to claim 2,wherein the light is fluorescence.
 4. The method according to claim 2,wherein the light is a chemical luminescence.
 5. The method according toclaim 1, wherein the steps (a-iv) to (d) further comprise: (a-iv)preparing separate regions on a substrate by fixing probes on a surfaceof the substrate, wherein the separate regions comprise: a first regioncontaining probes which provide a signal of a certain intensity onreaction with a reference nucleic acid having the wild-type sequence, asecond region containing probes which provide weaker signals on reactionwith the reference nucleic acid, and a third region containing probeswhich do not form hybrids on reaction with the reference nucleic acid;(b and c) reacting the DNA array of step (a) with the reference nucleicacid and measuring a signal of at least one region selected from thethree regions to obtain a first pattern; (b′ and c′) reacting the DNAarray of step (a) with the sample nucleic acid and measuring a signal ofat least one region corresponding to the at least one region selected instep (b and c) to obtain a second pattern; and (d) determining thepresence or absence of variation in the sample nucleic acid by comparingthe first and second patterns.
 6. The method according to claim 5,wherein the at least one region selected in step (b and c) is the firstregion giving a strongest total signal and/or the third region giving noor a weakest signal on reaction with the reference nucleic acid.
 7. Themethod according to claim 5, wherein the separate regions are arrangedon the substrate in order of signal intensity along a direction of adetection, wherein the signal intensity is obtainable on a reaction withthe reference nucleic acid.
 8. The method according to claim 5, whereinthe at least one region selected in step (b and c) is the third regionand the sample nucleic acid is determined to have variation when thesignal is detected in the third region with the sample nucleic acid instep (b′ and c′).
 9. The method according to claim 5, wherein the atleast one region selected in step (b and c) are both the first and thethird region and determining the presence or absence of variation isdetermined by comparing the ratio of the intensity of the third regionto that of the first region.
 10. The method according to claim 5,wherein all three regions are selected in step (b and c) and thepresence or absence of variation is determined by comparing thehistogram pattern of signal intensity.
 11. The method according to claim5, wherein detection of the total signal is performed by an area sensor.12. The method according to claim 7, wherein detection of the totalsignal is performed by a line sensor.
 13. The method according to claim1, wherein a base length of the probes is 8 to 30 nucleotides.
 14. Themethod according to claim 13, wherein the base length of the probes is12 to 25 nucleotides.